Date of Award

2001

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Terry W. Fenger

Second Advisor

Sheldon N. Finver

Third Advisor

Todd L. Green

Fourth Advisor

Vernon E. Reichenbecher

Fifth Advisor

Donald A. Primerano

Sixth Advisor

Leonard L. Deutsch

Abstract

Inducible nitric oxide synthase (iNOS) and manganese superoxide dismutase (MnSOD) are two enzymes that influence reactive oxygen species within the cell. The promoter regions for the genes encoding these enzymes have two specific transcription factor elements in common, activator protein 1 (AP-1) and nuclear factor kappa b (NF-KB). This study was initiated to determine if either NF-KB or AP-1 regulates the transcription of both genes in endothelial cells. To test whether NF-KB or AP-1 binding sites in the MnSOD and iNOS promoter region were important for transcriptional regulation, we performed induction analysis using various deletion constructs of MnSOD and iNOS luciferase reporter plasmids expressed in rat aortic endothelial cells (SVAREC and PRAEC). We found that the basal and inducible regulation of MnSOD are dependent upon the NF-KB binding site. Deletion of this ten base pair sequence produced a 90% decrease in basal expression and a 99% decrease in expression of lipopolysaccharide (LPS) treated cells, also studies with the inhibitors parthenolide and A-Fos, supported the finding that NF-KB is critical for MnSOD transcription. The AP-1 site is not necessary for basal transcriptional regulation of MnSOD but does seem to be necessary for induction of MnSOD, since deletion ofthe AP-1 binding site reduces reporter expression of LPS treated cells down to basal levels. Taken together, these results together suggest that MnSOD expression is dependent upon NF-KB and AP-1 for induction of transcription, while only NF-KB is necessary for basal levels of MnSOD transcription. Serial deletions and inhibitor studies of iNOS suggested that the NF-KB site between -680 and -266 is important for iNOS transcription. All of this data suggests that MnSOD and iNOS are reliant upon the transcription factor NF-KB for induction of transcription.

Subject(s)

Nitric oxide synthase.

Manganese enzymes.

Superoxide dismutase.

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